Growth Inhibition With Reversible Cell Cycle Arrest of Carcinoma Cells by Flavone L86-8275

Background: Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can inhibit tumor cell growth in vitro, and preliminary in vivo studies of the flavone L86-8275 have shown growth inhibition of LX529 and A549 lung carcinomas. L86-8275 [(-)cis-5, 7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-1-benzopyran-4-one] is a flavone of novel structure. Purpose: The purpose of this study was to determine in vitro whether L86-8275 is a more potent inhibitor of growth in breast carcinoma and lung carcinoma cells than quercetin or genistein. Methods: We studied the effects of L86-8275 on cell growth in seven breast carcinoma cell lines and five lung carcinoma cell lines. MDA468 breast carcinoma was then selected for further study. Cell proliferation was measured by a colorimetric dye reduction assay; synthesis of DNA, RNA, and protein by incorporation of the radioactive metabolic precursors thymidine, uridine, or leucine, respectively; adenosine triphosphate (ATP) content by a luciferase-mediated bioluminescence reaction; and cell cycle progression by the use of cell-synchronizing drugs (aphidicolin and nocodazole) and flow cytometry. Results: L86-8275 was not cytotoxic to stationary-phase cells but reversibly inhibited the growth of cells in exponential growth phase. At concentrations of 25-160 nM, L86-8275 inhibited growth of human breast and lung carcinoma cell lines by 50%. MDA468 breast carcinoma cells were 60-fold and 400-fold more sensitive to L86-8275 than to quercetin and genistein, respectively. By 24 hours after addition of L86-8275, DNA synthesis in MDA468 cells was inhibited by greater than 95%, protein synthesis by 80percnt;, and RNA synthesis by 40%-60%, under conditions that preserved cellular ATP levels at approximately 80%-90% of control values. When MDA468 cells released from aphidicolin-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed the S phase but arrested in G2. When cells released from nocodazole-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed mitosis but arrested in G1. Conclusions: L86-8275 is a potent, yet reversible, growth-inhibitory flavone that can selectively block cell cycle progression in vitro at more than one point in the cell cycle. Implications: These findings suggest that L86-8275 is a candidate for further preclinical development, as well as a model for the synthesis of other flavonoids that might potently delay cel